Potential Application of the
Oryza sativa
Monodehydroascorbate Reductase Gene
(
OsMDHAR
) to Improve the Stress Tolerance
and Fermentative Capacity of
Saccharomyces cerevisiae
Il-Sup Kim1☯*, Young-Saeng Kim2☯, Yul-Ho Kim3, Ae-Kyung Park4, Han-Woo Kim4, Jun-Hyuk Lee4, Ho-Sung Yoon1*
1School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea,2Division of Biological Sciences, University of California San Diego, La Jolla, California, United States of America,3Highland Agriculture Research Institute, National Institute of Crop Science, Rural Development Administration, Pyeongchang, Republic of Korea,4Division of Polar Life Sciences, Korea Polar Research Institute, Incheon, Republic of Korea
☯These authors contributed equally to this work. *92kis@hanmail.net(I-SK);hsy@knu.ac.kr(H-SY)
Abstract
Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is an important enzyme for ascor-bate recycling. To examine whether heterologous expression ofMDHARfromOryza sativa (OsMDHAR) can prevent the deleterious effects of unfavorable growth conditions, we con-structed a transgenic yeast strain harboring a recombinant plasmid carryingOsMDHAR (p426GPD::OsMDHAR).OsMDHAR-expressing yeast cells displayed enhanced tolerance to hydrogen peroxide by maintaining redox homoeostasis, proteostasis, and the ascorbate (AsA)-like pool following the accumulation of antioxidant enzymes and molecules, meta-bolic enzymes, and molecular chaperones and their cofactors, compared to wild-type (WT) cells carrying vector alone. The addition of exogenous AsA or its analogue isoascorbic acid increased the viability of WT andara2Δcells under oxidative stress. Furthermore, the sur-vival ofOsMDHAR-expressing cells was greater than that of WT cells when cells at mid-log growth phase were exposed to high concentrations of ethanol. HighOsMDHARexpression also improved the fermentative capacity of the yeast during glucose-based batch fermenta-tion at a standard cultivafermenta-tion temperature (30°C). The alcohol yield ofOsMDHAR -express-ing transgenic yeast dur-express-ing fermentation was approximately 25% (0.18 gg-1) higher than
that of WT yeast. Accordingly,OsMDHAR-expressing transgenic yeast showed prolonged survival during the environmental stresses produced during fermentation. These results suggest that heterologousOsMDHARexpression increases tolerance to reactive oxygen species-induced oxidative stress by improving cellular redox homeostasis and improves survival during fermentation, which enhances fermentative capacity.
a11111
OPEN ACCESS
Citation:Kim I-S, Kim Y-S, Kim Y-H, Park A-K, Kim H-W, Lee J-H, et al. (2016) Potential Application of theOryza sativaMonodehydroascorbate Reductase Gene (OsMDHAR) to Improve the Stress Tolerance and Fermentative Capacity ofSaccharomyces cerevisiae. PLoS ONE 11(7): e0158841. doi:10.1371/ journal.pone.0158841
Editor:Alvaro Galli, CNR, ITALY
Received:April 22, 2016
Accepted:June 22, 2016
Published:July 8, 2016
Copyright:© 2016 Kim et al. This is an open access article distributed under the terms of theCreative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability Statement:All relevant data are available from Dryad (doi:10.5061/dryad.9bv73).
Funding:This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ011122022016), Rural Development Administration, Korea.
Introduction
In aerobic organisms, oxygen is a double-edged sword; it is absolutely required for normal met-abolic growth, yet continuous exposure to oxygen can cause cellular damage. This is because molecular oxygen is continually reduced within cells to reactive oxygen species (ROS), such as superoxide anions (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH). In addi-tion, abiotic and biotic stimuli can lead to further ROS accumulation [1]. The produced ROS react readily with cellular components to generate acute or chronic damage that is sufficient to cause cell death, aging, and various disease [2]. Redox balance is maintained via the constitutive action of various antioxidant mechanisms that scavenge ROS, and both enzymatic and non-enzymatic processes can neutralize ROS [3].
Ascorbate (AsA)/D-erythroascorbate and glutathione are important antioxidants that are maintained in their reduced forms by enzymes in the AsA-glutathione cycle in higher eukary-otes, especially plants. The enzymes in this cycle, including AsA peroxidase (APX), monodehy-droascorbate reductase (MDHAR), dehymonodehy-droascorbate reductase (DHAR), and glutathione reductase (GR), are considered critical for minimizing and/or protecting cells from ROS induced by abiotic stressors [4]. MDHAR belongs to the flavoprotein family of pyridine nucle-otide-disulfide oxidoreductases, which includes thioredoxin reductase (Trr), lipoamide dehy-drogenase, GR, and mercuric ion reductase, and it catalyzes the reduction of
monodehydroascorbate (MDHA) and MDHA radicals to AsA using NAD(P)H as an electron donor [5]. Therefore, MDHAR plays an important role in maintaining the pool of AsA derived from MDHA. MDHAR is found in many eukaryotes, including cucumbers, potatoes, soybean root nodules, and rot fungus [6], and it is localized in chloroplasts, mitochondria, peroxisomes, and the cytosol in plants [7], and in microsomes, mitochondria, the Golgi apparatus, and eryth-rocytes in animals [8]. MDHAR cDNAs have been cloned from the following plants:Brassia rapa, cucumber, pea leaves, tomatoes, rice,Arabidopsis thaliana, and leaf mustard [9–12]. The MDHAR amino acid sequence is unique and it shares limited identity with other flavin oxido-reductases in eukaryotes [5]. Transcriptional activation of theMDHARgene has been reported under some environmental stresses, including oxidative stress such as H2O2, paraquat (PQ), and salicylic acid inB.rapa[13]; strong illumination in wheat leaves [14]; SO2and O3in coni-fer needles [15]; drought in grasses [16]; and internode rubbing in tomato plants [17].
Unlike in plants, little is known about the stress response involving MDHAR in yeast. In this study, a cDNA encoding the cytosolicMDHARfrom the rice plantOryza sativa( OsMD-HAR) was cloned, and its function was analyzed in a genetically modifiedS.cerevisiaestrain. Herein, we report that heterologousOsMDHARexpression improves tolerance to ROS-induced oxidative stress and fermentative capacity inS.cerevisiae. Furthermore, these findings improve our understanding of the development of ethanol-tolerant yeast strains and demon-strate the potential application of transgenic yeast in the fermentation field for industrial bioethanol production using glucose-based biomass.
Materials and Methods
Construction of
OsMDHAR
-expressing transgenic yeast
Total RNA was isolated from the leaves of 4-week-oldO.sativaseedlings, and the cDNA was synthesized by reverse transcription-polymerase chain reaction (RT-PCR). TheOsMDHAR
coding region was amplified from the cDNA by PCR usingTaqandPfupolymerases (Roche, Basel, Switzerland). The reaction conditions were as follows: initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min, and a final exten-sion at 72°C for 7 min. The primer set used for PCR cloning of theOsMDHARgene is shown inS1 Table. The PCR product was ligated into the pCR2.11TOPO-TA cloning vector (Invitro-gen, Carlsbad, CA, USA). After sequence confirmation and digestion withEcoRI, the DNA fragment containing theOsMDHARgene was ligated into the yeast expression vector p426GPD (Euroscarf, Frankfurt, Germany). The construct was transformed intoS.cerevisiae
BY4741 cells (S2 Table) and derivatives using the PEG/LiCl method [24]. Transformants were selected by plating cells on minimal agar medium (0.67% yeast nitrogen base without amino acids and with ammonium sulfate and 0.192% yeast synthetic drop-out medium lacking uracil, with 2% glucose, and 2% bacto agar) at 28°C for 3 days. Single colonies were restreaked, cul-tured under the same conditions, and then used for subsequent experiments.
Semi-quantitative RT-PCR
Total RNA from mid-log phase yeast cells was obtained using the Total RNA Isolation Kit (Promega, Madison, WI, USA). RT-PCR was performed using Maxime RT-PCR Premix (iNtRON Biotechnology Inc., Seongnam, Korea) according to the manufacturer’s instructions. The oligonucleotide primers used were OsMDHAR-F and OsMDHAR-R (S1 Table). PCR amplicons were resolved in 1.5% agarose gels, stained with ethidium bromide, and photo-graphed. ThePDA1amplicon was used as a reference [25].
Stress tolerance assay
exposed to 10 mM H2O2for 1 h at 28°C, serially diluted, and then spotted onto YPD agar plates.
Effect of exogenous antioxidants
Yeast cells in mid-log phase were treated with 10 mM AsA and 10 mM isoascorbate (IAA) for 1 h at 28°C, washed twice with cold phosphate-buffered saline (PBS) to remove residual antiox-idants, and suspended in fresh YPD medium. Resuspended cells were exposed to 20 mM H2O2 for 1 h at 28°C with shaking, appropriately diluted, and spotted onto YPD agar plates. For growth kinetics, cells (1 × 106cells/mL) grown at 28°C overnight were inoculated into YPD medium containing 10 mM AsA and 10 mM IAA in the absence or presence of 5 mM H2O2. The OD600was measured at 2-h intervals for the indicated time.
Protein preparation
Mid-log phase cells were incubated in the absence or presence of 20 mM H2O2for 1 h at 28°C and harvested by centrifugation at 4°C. Crude protein extracts were prepared using glass beads. Cells were washed three times with cold PBS and resuspended in lysis buffer (50 mM HEPES, pH 7.2, 5% glycerol, 1 mM PMSF, and protease inhibitor cocktail) with an equal volume of glass beads (425–600μm; Sigma-Aldrich, St. Louis, MO, USA). After vigorously vortexing 4 times for 1 min each at 2-min intervals on ice, the lysates were centrifuged at 13,000 ×gfor 20 min at 4°C, and the supernatants were used as protein extracts. Protein concentration was determined by using Protein Dye Reagent (Bio-Rad, Hercules, CA, USA).
MDHAR activity assay
MDHAR activity was assayed spectrophotometrically. The assay was performed at 25°C with a reaction mixture containing 50 mM potassium phosphate, pH 7.2, 0.2 mM NADH, 2 mM AsA, 1 unit of AsA oxidase (Sigma-Aldrich), and crude extract. MDHAR activity was mea-sured by monitoring the decrease in absorbance at 340 nm resulting from NADH oxidation. Activity was calculated using an absorbance coefficient of 6.2 mM−1cm−1. One unit is the
amount of enzyme that oxidizes 1 nmol of NADH per min at 25°C [26].
Western blot analysis
were used as previously described [28]. Anti-tubulin antibody (Tub; Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control. The anti-MDHAR antibody was produced in rabbits inoculated with purified MDHAR protein [29].
Measurement of AsA-like content
Yeast cells were treated with or without 20 mM H2O2for 1 h at 28°C, harvested by centrifuga-tion, and washed twice with cold PBS. AsA-like content was determined spectrophotometri-cally. Yeast cells were disrupted with glass beads and 5%m-phosphoric acid. The homogenate was centrifuged at 12,000 ×gfor 20 min. Total AsA content was determined in a reaction mix-ture containing 100μL of supernatant, 500μL of 150 mM KH2PO4buffer (pH 7.4) containing 5 mM EDTA, and 100μL of 10 mM dithiothreitol (DTT) to reduce dehydroascorbate [30] to AsA. The reaction mixtures were incubated for 10 min at room temperature. Then, 100μL of 0.5% N-ethylmaleimide (NEM) was added to remove excess DTT. Ascorbic acid was assayed in a similar manner, except that 200μL of deionized H2O was substituted for DTT and NEM. In both reactions, color was developed by adding 400μL of 10% trichloroacetic acid, 400μL of 44%o-phosphoric acid, 400μL ofα,α0-dipyridyl in 70% ethanol, and 200μL of 30 gL-1FeCl3. The reaction mixtures were incubated at 40°C for 1 h and quantified spectrophotometrically at 525 nm. The concentration of DHA was estimated from the difference in the concentrations of total and reduced AsA. The concentrations of AsA and DHA were quantified by generating of a standard curve using AsA from 1 to 10μM mL-1[31].
Cellular redox state assay
Cells were cultured for 6 h at 28°C until they reached mid-log phase and then exposed to 20 mM H2O2for 1 h at 28°C. The cells were harvested and lysed with glass beads. The intracellular hydroperoxide level was determined by ferrous ion oxidation in the presence of the ferric ion indicator xylenol orange [32]. Fifty microliters of crude cell extract were added to 950μL of FOX reagent (100μM xylenol orange [water-soluble form; Sigma-Aldrich], 250μM ammo-nium ferrous sulfate, 100 mM sorbitol, and 25 mM sulfuric acid). The mixture was incubated at room temperature for 30 min and then centrifuged to remove any flocculated material before measuring the absorbance at 560 nm. Hydroperoxide concentration is reported asμg per mg of protein. For the fluorescence assay, yeast cells were treated with 0.1 mM dichlorodihydrofluor-escein diacetate (DCFHDA) and 0.1 mM dihydrorhodamine 123 (DHR 123) for 20 min at 28°C with shaking before treatment with 10 mM or 20 mM H2O2for 1 h at 28°C. Treated cells were washed twice with PBS and visualized by fluorescence microscopy (excitation, 488 nm; emission, 525 nm) [33]. Protein oxidation was determined using the Protein Carbonyl Content Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol.
Laboratory glucose-based batch fermentation
Cells (initial concentration adjusted to 1 × 106cells per mL) grown at 30°C overnight were inoculated into YG medium containing 20% glucose and 1% yeast extract and then fermented under aerobic conditions on a shaker (160 rpm) for 72 h at 30°C. The alcohol concentration was determined based on the percentage (v/v) of alcohol in the distillate after fermentation, which was measured using an alcohol hydrometer (REF 503; Korins, Seoul, Korea). The resid-ual glucose concentration was measured with a hand-held Refractometer N1 (Atago, Tokyo, Japan). Alcohol and residual glucose were measured after removing cell debris by centrifuga-tion (2,000 ×g, 3 min). Growth was monitored for 24 h under the same conditions. To investi-gate cell survival during fermentation, cells were harvested at three time points (24, 48, and 72 h) and serially diluted to 10−9with deionized distilled water, and then 5
plated onto YPD agar, incubated for 3 days at 30°C, and then photographed. During fermenta-tion, MDHAR expression was analyzed at various time points by western blotting as described above.
Statistical analysis
Significant differences in the measured parameters were identified using Origin Pro 8.0. Means were considered to be significantly different whenP<0.05. All experiments were
indepen-dently performed at least three times, and the results are expressed as the mean ± standard deviation (SD). The results of the spotting, growth kinetics, and fermentation assays are repre-sentative of at least two independent experiments performed under identical conditions.
Results
Generation of
OsMDHAR
-expressing recombinant yeast and their stress
response to an oxidant
A cDNA containing the open reading frame (ORF) ofMDHARfromO.sativa(OsMDHAR) was subcloned into the yeast expression vector p426GPD, which allows constitutive expression of a target gene under the control of the yeastGPD(glyceraldehyde-3-phosphate dehydroge-nase) promoter (Fig 1A). To determine whether theOsMDHARgene is expressed in yeast, semi-quantitative RT-PCR and immunoblotting were performed. A single DNA fragment of approximately 494 bp corresponding to a region withinOsMDHARwas amplified from cells transformed with the p426GPD-OsMDHARconstruct (TC), whereas no amplification signal was detected in wild-type cells transformed with vector alone (Fig 1B). To investigate whether the OsMDHAR protein is expressed, western blotting and enzymatic activity assays were per-formed. As shown inFig 1C, immunoblotting analysis using an antibody raised against MDHAR showed a single band at 47 kDa derived from theMDHARtransgene in the crude extract prepared from the TC cells, but not in extract prepared from WT cells. The enzymatic activity of MDHAR in the TC cells was approximately 10-fold higher than that in the WT cells (Fig 1D). There was a clear correlation between theOsMDHARmRNA level and the MDHAR protein level. To determine ifOsMDHARis related to stress tolerance in yeast, a cell survival assay was used. In this assay, the optical density of the TC cells was higher than that of the WT cells at a variety of H2O2concentrations. The WT cells were completely inhibited in 8 mM H2O2, whereas the TC cells were inhibited in 10 mM H2O2(Fig 1E). To confirm these results, growth kinetics, streaking, and spotting assays were performed. In the growth kinetics assay, the TC cells recovered more rapidly than the WT cells did in the presence of 5 mM H2O2for the indicated times. However, under normal conditions, there was no difference between the cells (Fig 1F). The streaking assay results supported the growth rate findings (Fig 1F, boxed panel). The spotting assay also demonstrated that the TC cells had acquired increased stress tolerance when they were exposed to 20 mM H2O2for 1 h at 28°C with shaking, compared to that of the WT cells (Fig 1G). These results suggest thatOsMDHARexpressed inS.cerevisiae
as a eukaryotic model system effectively enhances stress tolerance when the cells are challenged with oxidative stress.
Expression of cell rescue proteins and the redox state under oxidative
stress
to oxidative stress. A wide range of proteins was upregulated in TC cells during oxidant chal-lenge compared to the expression levels in WT cells (Fig 2). The expression of several metabolic enzymes, including Hxk, GAPDH, G6PDH, Ald, and Adh, was increased in the TC cells under oxidative stress, compared to that in WT cells. In addition, numerous antioxidant enzymes were upregulated, including Sod1, Gpx, GR, Trx2, Trx3, Tsa1, and Por, in TC cells. However, Fig 1. Construction of anOsMDHAR-expressing yeast vector and the stress response ofOsMDHAR-expressing yeast to hydrogen peroxide. (A) Schematic diagram of the p426GPD::OsMDHAR construct. TheOsMDHARgene (approximately 1.5 kbp) was subcloned to generate the p426GPD:: OsMDHAR construct withOsMDHARunder the control of the constitutiveGPDpromoter. Semi-quantitative RT-PCR (B), immunoblotting (C), and an enzymatic assay (D) were performed to examine whetherOsMDHARis expressed in this yeast strain.PDA1and tubulin (Tub) were used as
housekeeping controls for RT-PCR and western blotting, respectively. The molecular size of the PCR product and molecular weight of the detected band were approximately 494 bp and 47 kDa, respectively. Stress tolerance to hydrogen peroxide was evaluated by cell survival, growth kinetics, and spotting assays. (E) To monitor cell viability, yeast cells precultured in YPD medium were inoculated into fresh YPD medium and exposed to different
concentrations of H2O2for 16 h at 28°C. Then, the optical density at 600 nm (OD600) was measured. Circles, cells transformed with p426GPD-OsMDHAR
(TC cells); squares, wild-type (WT) cells transformed with an empty vector. (F) For the growth kinetics assay, precultured yeast cells were inoculated into YPD medium containing 5 mM H2O2, and the OD600was measured at 2-h intervals for 36 h. A streaking assay was also performed, in which mid-log
phase yeast cells (OD6002.0) were streaked onto YPD agar plates supplemented with 5 mM H2O2. WT (squares) and TC (circles) cells in the absence
of 5 mM H2O2; WT (upward triangles) and TC (diamonds) cells in the presence of 5 mM H2O2. (G) Mid-log phase yeast cells were exposed to 20 mM H2O2
for 1 h with shaking, and serially diluted with YPD medium. A 5-μL aliquot of each dilution was spotted onto YPD agar plates.
cytosolic Trr1 expression was similar in TC and WT cells grown under oxidative stress (Fig 2A). High expression of metabolic enzymes and antioxidant enzymes resulted in improved redox homeostasis in TC cells. The hydroperoxide level in TC cells was approximately 1.8-fold higher than that in WT cells cultured in the presence of H2O2; however, there was no difference between TC and WT cells cultured under normal conditions (Fig 2B). These results were sup-ported by DCFHDA fluorescence, an indicator of cytosolic hydroperoxide, as DCFHDA fluo-rescence was lower in TC cells than in WT cells, although there was a change in signal intensity in both cells under oxidative stress (Fig 2C). Expression of Sod1, Tsa1, Por1, and Por2 was Fig 2. Analyses of cell rescue proteins, redox state, and protein oxidation under oxidative conditions.(A) Expression changes in antioxidant and metabolic enzymes in mid-log phase yeast cells exposed to 20 mM H2O2for 1 h with shaking. Tubulin (Tub) was used as a loading control. (B)
Hydroperoxide levels in TC cells in the absence (red bar) and presence (green bar) of 20 mM H2O2were assessed using FOX reagent and were
calculated relative to that in WT cells grown under normal conditions, which was set to 100%. (C) Mid-log phase yeast were exposed to 20 mM H2O2for 1
h at 28°C with shaking. Redox state was analyzed by measuring DCFHDA oxidation as an indicator of cytosolic ROS. (D) Sensitivity of mutants (sod1Δ,
tsa1Δ,por1Δ, andpor2Δ) to oxidative stress. Yeast cells (OD6001.0) were exposed to 10 mM H2O2for 1 h at 28°C with shaking, serially diluted with
YPD medium, spotted onto YPD agar plates, and incubated for 2–3 days. (E) Expression changes in molecular chaperones in mid-log phase yeast cells
exposed to 20 mM H2O2for 1 h with shaking. Tubulin (Tub) was used as a loading control. (F) Protein carbonylation in yeast cells exposed to 20 mM H2O2
for 1 h was calculated relative to that in WT cells under normal conditions, which was set to 100%. Red bar, normal conditions; green bar, H2O2treatment;
WT, yeast cells with an empty vector; TC,OsMDHAR-expressing yeast cells; N, normal conditions; S, H2O2treatment.
inhibited or unchanged in WT cells under oxidative stress, compared to the expression levels in TC cells. Mutants with deletions in antioxidant enzymes (sod1Δ,tsa1Δ,por1Δ,por2Δ, and
ara2Δ) were hypersensitive to oxidative stress (Fig 2D). Unexpectedly, the recovery ofpor1Δ cells in growth kinetics and spotting assays was slower than that of WT cells without an empty vector (BY cells) in the presence of H2O2; however, there was no difference between these cells under normal condition (S1A and S1B Fig).Por1Δcells displayed increased cytosolic and mito-chondrial hydroperoxide levels under stress compared to the levels in BY cells (S1C Fig). In addition,por1Δcells were sensitive to a wide range of stressors, including oxidants (menadione [MD] andtert-butylhydroperoxide [t-BOOH]), heat shock, metals (copper, iron, and zinc), heavy metals (aluminum and cobalt), acids (sulfuric acid and salicylic acid), and high salinity (NaCl) (S1D Fig). The expression of various molecular chaperones, HSPs, and cofactors was increased in TC cells under oxidative stress. The highly expressed proteins included Hsp104, Hsp82, Hsp60, Hsp30, Hsp26, and two members of the Hsp70 family (Ssa1 and Ssb1).
Although the expression of Hsp42 in TC cells was lower under oxidative stress than under nor-mal conditions, it was still higher in TC cells than in WT cells (Fig 2E). This high expression of chaperone proteins in TC cells resulted in decreased protein oxidation, and the protein car-bonyl content of TC cells was lower than that of WT cells (Fig 2F). Thus, TC cells express a range of rescue proteins that enable them to respond to oxidative stress by improving redox homeostasis and proteostasis following the decreases in cellular hydroperoxide and protein oxidation levels.
AsA-like content and its effect on H
2O
2insufficient to promote stress tolerance, compared to the levels in TC cells. Based on these results, in subsequent experiments, we tested the effects of exogenous AsA or its analogue IAA on the stress response of yeast cells. Although WT cells were more sensitive to oxidative stress than TC cells, spotting assays showed that WT cells reached the same growth levels as TC cells in the presence of exogenous AsA or IAA, which is indicative of adaptation capacity under oxi-dative stress (S2A Fig, upper panel). Supplementation with these molecules led to enhanced Fig 3. Stress response related to ascorbate (AsA)-like molecules.(A) AsA-like content in yeast cells exposed to 20 mM H2O2for 1 h was analyzed
and is shown as nmol per mg protein. The ratio shown is that of the reduced form to oxidized form. (B)ARA2expression was evaluated by
semi-quantitative RT-PCR.PDA1was used as a control. (C) Oxidative stress response of yeast cells in the absence and presence ofARA2. Mid-log phase cells were serially diluted, and 5μL of the diluted solutions were spotted onto YPD agar plates containing 4 mM H2O2(upper panels). Mid-log phase cells were
treated with 20 mM H2O2for 1 h with shaking, diluted with YPD medium, and spotted onto YPD agar plates. The plates were incubated for 2–3 days and
photographed. (D) Stress sensitivity ofara2Δyeast cells, in which the erythroascorbate (EAA) biosynthesis gene was deleted. Yeast cells (A6001.0)
were exposed to 10 mM H2O2for 1 h at 28°C with shaking, serially diluted with YPD medium, spotted onto YPD agar plates, and incubated for 2–3 days.
(E) The redox state ofara2Δyeast cells under oxidative conditions. Yeast cells (OD6001.0) were exposed to 10 mM H2O2for 1 h after DCFHDA and
DHAR 123 treatment for 30 min and washed twice with phosphate-buffered saline (PBS). Probe intensity was observed by fluorescence microscopy. BY, wild-type cells without an empty vector;ara2Δ, cells with a deletion of the EAA biosynthetic geneARA2; WT, wild-type yeast cells with an empty vector; TC, yeast cells with p426GPD::OsMDHAR; WA,ara2Δyeast cells with an empty vector; TA,ara2Δyeast cells with p426GPD::OsMDHAR; N, normal conditions; S, in the presence of H2O2.
stress tolerance inara2Δcells under oxidative stress (S2A Fig, lower panel).Ara2Δcells were also sensitive to various stressors, including heat shock, MD,t-BOOH, ethanol, NaCl, cad-mium, zinc, and lactic acid (S2B Fig).
Effect of
OsMDHAR
expression under batch fermentation
Since TC cells expressingOsMDHARwere more tolerant to oxidative stress than WT cells, these cells were tested in glucose-based batch fermentation in YG medium under aerobic con-ditions. Distinct differences in alcohol yield and residual glucose content were observed between TC and WT cells at 30°C, which is the temperature that is typically used for industrial fermentation. During fermentation for 72 h at 30°C, the alcohol yield of TC cells was approxi-mately 25% higher than that of WT cells. The final alcohol concentration was approxiapproxi-mately 13.5% and 10.2% with TC cells and WT cells, respectively, and the residual glucose concentra-tion was inversely proporconcentra-tional to the alcohol concentraconcentra-tion during fermentaconcentra-tion (Fig 4A).
OsMDHARexpression in the TC cells during fermentation was confirmed by western blotting, although protein expression decreased slightly over time (Fig 4B). In addition, distinct difences in growth kinetics and cell survival were observed between TC and WT cells during fer-mentation. The growth rate in early fermentation until stationary phase was higher in TC cells than in WT cells (Fig 4C). In particular, the survival of TC cells was time-dependently higher than that of WT cells during fermentation in YG medium for 72 h, although cell viability decreased gradually with time (Fig 4D). Next, to examine the response to various concentra-tions of ethanol, yeast cells were grown to log phase (OD600= 2.0), and then exposed to 15% or 20% ethanol for 1 h with shaking. After exposure, the cultures were serially diluted and spotted onto YPD agar plates. TC cells were more tolerant to ethanol than WT cells (Fig 4E). In addi-tion, TC cells expressingOsMDHARwere more resistant to ROS-induced oxidative stress (MD,t-BOOH, copper, iron, cadmium, and sodium dodecyl sulfate [SDS]) than WT cells (S3 Fig). Therefore, our findings indicate thatOsMDHARexpression enhances fermentative capac-ity at moderate temperatures, which is an important factor for increased alcohol yield during fermentation.
Discussion
We evaluated anOsMDHAR-expressing yeast strain and compared it a control WT strain to determine ifOsMDHARcould improve the stress tolerance ofS.cerevisiaeas a eukaryotic model system. The viability of cells transformed with p426GPD::OsMDHAR (TC cells), which constitutively expressOsMDHARunder the control of theGPDpromoter, was inhibited by a higher concentration of H2O2, than the concentration that inhibited the viability of WT cells with an empty vector (Fig 1). In growth kinetics, streaking, and spotting assays,OsMDHAR -expressing TC cells were more resistant to H2O2than WT cells. We measured the stress toler-ance of TC cells containing recombinantOsMDHARin the presence of 5 mM or 20 mM H2O2.
OsMDHARexpression in yeast enhanced the acquired resistance to H2O2(Fig 1) and to a wide range of ROS-induced oxidative stressors, including oxidants (MD andt-BOOH), redox-active metals (iron, copper, and cobalt), a non-redox-active metal (cadmium), SDS, and ethanol (S3 Fig). Although no previous studies have established a relationship betweenOsMDHAR expres-sion and the stress response in yeast, the overexpresexpres-sion of eukaryotic antioxidant genes has been shown to improve stress resistance in both prokaryotes and eukaryotes [35–37].
To investigate whether the improved tolerance to oxidative stress in TC cells was due to an
and Por (Fig 2A). In contrast, Trr1 expression was the same in TC and WT cells under stress. The expression of most proteins was dependently induced in TC cells. The major stress-protection mechanisms include synthesis of antioxidant proteins, proteins involved in meta-bolic and pentose phosphate pathways and molecular chaperones, and the accumulation of compatible solutes and hydrophylins as well as permeability adaptation of the plasma mem-brane [18]. Upregulation or overexpression of stress-related genes (SOD,CAT,GPX,TSA1, Fig 4. Fermentative capacity and the survival ofOsMDHAR-expressing yeast cells during batch fermentation.(A) Fermentative capacity was analyzed by measuring the alcohol (AC) and residual glucose (RG) concentrations in YG medium after fermentation for 72 h at 30°C. Upward triangles, AC of WT cells; circles, AC of TC cells; squares, RG of WT cells; diamonds, RG of TC cells. (B) Time-dependentOsMDHARexpression during batch fermentation was evaluated by western blotting. Tubulin (Tub) was used as a loading control. (C) Growth kinetics during fermentation was assessed by measuring the OD600at 2-h intervals for the indicated time. Squares, WT cells; circles, TC cells. (D) Cell viability during fermentation at 30°C was
assessed by a spotting assay. Cells were harvested after 24 h (upper panel), 48 h (middle panel), and 72 h (lower panel) of fermentation and serially diluted to 10−9. A
5-μL aliquot of each diluted solution was spotted onto YPD agar plates. After incubation for 3 days, the plates were photographed. (E) Stress response to ethanol. Mid-log phase yeast cells (OD6002.0) were exposed to different concentrations of ethanol (0, 15%, and 20%) for 1 h,
serially diluted, and spotted onto YPD agar plates. WT, yeast cells with an empty vector; TC,OsMDHAR-expressing yeast cells.
andTRX) enhances acquired resistance to ROS-induced oxidative stresses, including oxidants (O2−, H2O2, and MD), ethanol, and lactic acid, by improving redox homeostasis following the reduction of oxidative damage through cross-protection between target proteins and other antioxidant enzymes inS.cerevisiae[38–42], whereas the direct interactions between H2O2 and other antioxidant proteins play a secondary role by protecting ribosomal proteins from stress-induced aggregation through the function of CAT and other peroxiredoxins [43,44]. In addition, the TRX system (TRX plus TRR) can act as an alternative system to reduce GSH disulfide (GSSG) in the presence of NADPH inglr1-deficient yeast cells and buffer oxidative stress [45]. TomatoMDHAR- andArabidopsis thalianaMDHAR (AtMDHAR1 )-overexpres-sing transgenic plants showed acquired tolerance against abiotic stressors (chilling and high temperature stresses, ozone, salt, polyethylene glycol, and radiation) by improving redox homeostasis through lowering the levels of hydrogen peroxide and lipid peroxidation and increasing the net photosynthetic rate, PSII effective quantum yield (Fv/Fm), fresh weight, and antioxidant’s enzyme activities (DHAR, GR, SOD and peroxidases) by elevating the AsA level compared to in WT plants [11,12,26]. In contrast, downregulation or repression of these anti-oxidant systems leads to a number of stress-dependent phenotypes, including genome instabil-ity [46], slow growth, and increased sensitivity to ROS-generating agents such as H2O2, which accelerates aging, auxotrophy, and cell death [47]. In addition, the suppression of proteins involved in the pentose phosphate pathway (i.e., G6PDH) and another pathway (isocitrate dehydrogenase) important for reducing power production increased the sensitivity to oxidative stress following an imbalance in NADPH homeostasis [47–49]. Furthermore, TC cells showed increased porin (Por) expression under oxidative stress (Fig 2A). Mitochondrial function affects redox homeostasis, which is mediated primarily by the voltage-dependent anion chan-nel (VDAC; porin pore). VDAC releases superoxide anions from the mitochondria into the cytosol and increases cytosolic ROS [50]. However,por1Δcells showed increased cytosolic and mitochondrial ROS levels (S1 Fig). Taken together, these findings highlight the importance of
OsMDHAR-mediated mechanisms in maintaining ROS at non-toxic levels and cross-protec-tion through the activacross-protec-tion of various antioxidant enzymes.
chaperone machinery systems, thus improving proteostasis under oxidative stress. However, the relationship betweenOsMDHARand Hsp expression remains unclear.
OsMDHARexpression in TC cells increased the ratio of reduced and oxidized forms follow-ing the accumulation of the AsA-like molecule EAA under oxidative stress (Fig 3A). TC cells were more tolerant to ROS-induced oxidative stress than WT cells (Fig 3C). These results show that EAA in WT cells is insufficient to induce the necessary machinery for stress tolerance compared to TC cells. Treatment of WT cells with AsA or its analogue IAA improved the stress response, such that it was similar to that of TC cells. Supplementation with AsA or IAA also enhanced acquired tolerance in WA cells, although the stress resistance of WA cells was lower than that of TC and WT cells (S2 Fig). These results show that EAA as an AsA analogue plays an important role in tolerance to oxidative stress. From this perspective, the increase in the AsA-like pool is mainly the result of EAA recycling through OsMDHAR as well as EAA bio-synthesis following upregulation ofARA2under unfavorable conditions. MDHAR, which is widely found in eukaryotes, is an important antioxidant that functions as a key enzyme for maintaining AsA pools. AsA is synthesized by all higher plants and nearly all higher animals, with the exception of some species, including humans, primates, guinea pigs, some birds, and fish [57]. AsA and its analogue EAA are the most abundant water-soluble free radical scaven-ger antioxidants in living organisms. AsA serves as a cofactor for enzymes involved in hormone biosynthesis and is involved in the recycling of antioxidants such as AsA,α-tocopherol, and phenolics [49]. The precise mechanisms underlying these functions are not yet known. In Can-dida albicans, EAA peroxidase is capable of detoxifying H2O2by converting it to water through oxidation of EAA, which increased tolerance to oxidative stress [34]. After participating in the reaction, EAA can be recycled by several different mechanisms. The EAA radical, produced fol-lowing EAA oxidation, can be recycled folfol-lowing reduction by OsMDHAR or mitochondrial NADH-dependent cytochrome b5reductase [58]. In bioassays using tobacco hornworm (
Man-duca sexta), EAA induced larval growth almost as well as AsA because AsA can be converted to EAA by a ubiquitin-mediated process [59]. Because of this OsMDHAR-mediated recycling, EAA content and its redox state are maintained, which is critical under ROS-induced oxidative stress. Additionally, EAA plays a role in cell growth and development, as well as in response to oxidative stress.
To further elucidate the functions ofOsMDHAR, the glucose-based batch fermentative capacity of TC and WT cells was compared at 30°C (a typical industrial temperature) because Brazil, the United States, and Korea produced approximately 21.6, 50.3, and 0.15 billion gallons of bioethanol, respectively in 2012, which is more than double that produced in 2007 [60]. Dur-ing batch fermentation at 30°C,OsMDHARwas constitutively expressed until the end of fer-mentation (Fig 2B). The alcohol concentrations produced by fermentation using TC and WT cells were 13.5% (0.71 ± 0.02 gg−1) and 10.2% (0.53 ± 0.03 g
g−1), respectively (Fig 4A), and
the alcohol concentration in TC cells was approximately 25% (0.18 gg−1) higher than that in
WT cells. In general, the theoretical ethanol yield from glucose fermentation byS.cerevisiaeis 10–11% (0.50–0.55 gg−1) [61]. Interestingly, the alcohol yield of TC cells in this study was
9.4% (0.24 gg−1) higher than of WT cells reported previously [61]. Survival during
fermenta-tion at 30°C was also higher for TC cells than for WT cells (Fig 4D). The alcohol concentration ofOsMDHAR-expressing TG yeast (13.5%; 0.71 ± 0.02 gg−1) was higher than that ofCaDHN
-(13.3%; 0.63 ± 0.03 gg−1) andOsTPX-expressing TG yeasts (12.9%; 0.61 ± 0.01 g
g−1); which
have some limitations, such as low ethanol yield and poor ethanol tolerance in typical glucose-based fermentation [64,65].Saccharomycesspp. is most attractive microorganism for ferment-ing sugars to ethanol. Besides its innate properties, overexpression of stress-related genes could be a useful method for developing ethanol-tolerant yeasts because ethanol toxicity is highly related to ROS generation. For example, overexpression ofTRX2increased fermentation capacity and wine yield and quality by preventing the oxidative damage to glycolytic and fermentation proteins (Ahp1) through cross-protection of transcription factor (Yap1) and antioxidant enzymes (Sod1, Sod2, and CAT) in industrial wine yeast during biomass-based batch- and fed-batch fermentation [42,66]. Upregulation ofG6PDH(ZWF1) [36],MPR1[67], andPAD[68] conferred tolerance to ethanol by enhancing NADPH and redox homeostasis or by detoxifying inhibitory compounds. To obtain a high ethanol yield in fermentation, improvedS.cerevisiaestrains that are tolerant of ethanol and different metabolic environments are required, such as theOsMDHAR-expressing transgenic yeast developed herein. As shown,OsMDHARexpression in TC cells increased toler-ance to ethanol and sugar-induced osmotic and physiochemical stresses.
In conclusion, high expression ofOsMDHARin a transgenic yeast strain increased acquired tolerance to ROS-induced oxidative stress, such as that generated by H2O2, by maintaining bal-anced cellular redox homeostasis, proteostasis, and the pool of EAA as an AsA-like molecule. It is possible that the expression ofOsMDHARin yeast enhanced tolerance to oxidative stress and that all antioxidant defense mechanisms act synergistically, cross-protecting the cells at different levels and functioning to generate a fast and effective defense response under oxida-tive stress. In addition, heterologousOsMDHARexpression enhanced tolerance to high con-centrations of ethanol, resulting in improved fermentative capacity. The genetically modified yeast used in this study show potential for use in the fermentation industry to improve ethanol yield. Further studies are needed to elucidate the mechanism underlying increased ethanol tol-erance and yield inOsMDHAR-expressingS.cerevisiaeduring fermentation.
Supporting Information
S1 Fig. Stress sensitivity and redox state ofpor14yeast cells under oxidative stress.
(DOCX)
S2 Fig. Exogenous effect of ascorbate and its analogue on the stress sensitivity ofara2Δ
yeast cells.
(DOCX)
S3 Fig. Response ofOsMDHAR-expressing yeast cells to abiotic stressors.
(DOCX)
S1 Methods. Cellular response and redox state inpor1Δyeast cells under oxidative stress.
(DOCX)
S2 Methods. Cellular response and redox state inara24yeast cells under oxidative stress and exogenous effect of AsA and its analogue.
(DOCX)
S3 Methods. Cellular response and redox state in yeast under oxidative stress.
(DOCX)
S1 Table. Oligonucleotide sequence used in this study.
(DOCX)
S2 Table. Strains and plasmids used in this study.
Author Contributions
Conceived and designed the experiments: I-SK H-SY. Performed the experiments: I-SK Y-SK A-KP H-WK J-HL. Analyzed the data: I-SK Y-SK. Contributed reagents/materials/analysis tools: Y-HK H-WK. Wrote the paper: Y-SK I-SK H-SY. Performed fluorescence image analysis and stress response assay: I-SK.
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